Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR TREATING AND REDUCING THE INCIDENCE OF
NEWCASTLE DISEASE
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates methods for treating or reducing the incidence
of
Newcastle disease. More particularly, the present invention relates to methods
for
treating, or reducing the incidence of Newcastle disease in birds as well as
reducing
the transmissivity of Newcastle disease.
2. Description of the Related Technology
Newcastle disease is an acute, febrile and highly contagious disease that
affects the respiratory and nervous systems of birds. Highly contagious and
easily
transmitted, the disease has been the cause of numerous epidemics in birds
since
1926. The disease generally appears suddenly and spreads quickly in
susceptible
flocks through direct contact with the secretions, feces, respiratory
discharges and
carcass, frozen or otherwise, of infected fowl or previously vaccinated fowl.
The
disease is also transmitted by contaminated surfaces, such as feed, water,
implements
or premises. The disease may result in asymptomatic infection to heavy
mortality. In
younger birds, mortality may range from a low percentage to 100%. Similarly,
mortality in laying flocks varies from 0 to 100% depending on the viral
strain. To
date, there is no known treatment or cure for Newcastle disease.
Newcastle disease is a paramyxovirus belonging to the group of myxoviruses
and has many different viral strains and forms. All strains of Newcastle
disease are
morphologically, structurally and serologically indistinguishable. However,
large
differences exist in the virulence of different strains for chickens, eggs and
tissue
culture systems. These differences are expressed in the classification of the
different
strains as velogenic, mesogenic and lentogenic strains.
Lentogenic strains, apathogenic, are used in the preparations of most live
vaccines. Lentogenic Newcastle disease is caused by mildly pathogenic strains
of
Newcastle disease virus and is characterized mainly by respiratory problems.
This
form of the disease is commonplace in the commercial poultry industry and
results in
economic losses due to livestock loss, poor livestock growth, feed conversion,
and
increased livestock carcass condemnation at processing.
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Mesogenic strains, which is intermediate in pathogenicity, are used as
vaccinal
strains for boosting the immunity of older fowls, e.g. in such commercially
available
mesogenic strains as Komarov (Haifa) and Roakin. Mesogenic Newcastle disease
is
typically found throughout the world in various fowl.
Velogenic strains are highly pathogenic and are used to test immunity and
mortality. Velogenic viscerotropic Newcastle disease is characterized by high
mortality with severe lesions in the gastrointestinal tract. Although present
in most
other areas of the world, it is not found in the United States. The last
outbreak of
velogenic viscerotropic Newcastle disease occurred in California during 1970-
74,
costing the United States Department of Agriculture (USDA) more than 60
million
dollars to eradicate the disease. Velogenic neurotropic Newcastle disease is
also
characterized by a high mortality and severe neurological symptoms. Although
seldom seen in the United States, Velogenic neurotropic Newcastle disease is
relatively widespread in other parts of the world.
In an effort to curtail the economic losses due to Newcastle disease in the
commercial poultry industry, young chickens have been routinely vaccinated
against
the Newcastle disease virus. The vaccines used are prepared with live
attenuated
Newcastle disease virus derived from lentogenic or mesogenic strains and
confer
immunity against all forms of Newcastle disease.
Chickens are typically inoculated by mass administration procedures including
the distribution of the live virus vaccine in drinking water and the spraying
of the
vaccine directly onto the chickens. Once inside the chicken, the virus
replicates in the
respiratory tract and is spread from chicken to chicken by aerosol and direct
contact
routes, theoretically immunizing a flock within to the various forms of
Newcastle
disease within a relatively short amount of time. This immunization, however,
frequently induces a subclinical or mild clinical form of Newcastle disease.
Any
conferred immunity tends to be non-uniform among the flock and of short
duration.
Inactivated vaccines are also inadequate, requiring the use of large amounts
of antigen
to induce immunity that is inefficiently administered parenterally and may
contain
allergenic substances, i.e. antibiotics, preservatives, etc. Moreover, because
the
immune response increases as the pathogenicity of the live vaccine increases,
immunization programs often involve sequential administration of multiple and
progressively more virulent viruses or live virus followed by administration
of
vaccines using inactivated viruses. Another significant problem is that some
virulent
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strains, such as the exotic Newcastle disease strain, are capable of infecting
and
killing birds that have been previously vaccinated.
As a result of these common problems, investigations have been conducted to
develop non-virus based treatment methods. Although there are some
publications,
such as U.S. publications nos. 2003/0185912 and 2005/0147697, that address the
antiviral properties of a composition composed of ginger, green tea, turmeric
and
horseradish, such publications do not disclose a method for treating Newcastle
disease.
Therefore, a need exists for methods of treating, reducing the incidence of,
or
reducing the transmissmivity of Newcastle disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a graph of dilution log versus cell concentration depicting the
affect of Composition 1 on the viability of Newcastle disease virus (NDV)
strain
B 1/B 1 in VERO E6 cells
Figure 2 shows a graph of dilution log versus cell concentration depicting the
affect of a composition in accordance with the invention on the viability of
NDV
strain B 1/B 1 in embryonating eggs.
Figure 3 shows on the left panel, cells showing no cytopathic effects (CPE)
following exposure to NDV treated with a 1x10-3 dilution of a composition in
accordance with the present invention, and on the right panel, cells with CPE
following exposure to NDV treated with a 1x10-3 dilution of the placebo.
SUMMARY OF THE INVENTION
The invention is directed to methods for the treatment of Newcastle disease,
for reducing the incidence of contracting Newcastle disease and for reducing
the
transmissivity of Newcastle disease.
In the first aspect of the invention, relates to a method for the prophylactic
use
of a composition to reduce the incidence of contracting and/or transmitting
Newcastle
disease. The method comprises the steps of administering to a bird that has
been,
might be or will be, exposed to the Newcastle virus, an amount of a
composition
having a first ingredient obtainable from turmeric extract; a second
ingredient
obtainable from green tea; and an acceptable cariier. The amount of anti-
microbial
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composition is effective, when administered, to reduce the incidence of
contracting
and/or transmitting Newcastle disease.
In a second aspect of the invention, the composition may be used to treat a
bird infected with Newcastle disease by administering an effective amount of
the
composition to treat Newcastle disease.
A third aspect of the invention relates to aerosol or liquid compositions
including having a first ingredient obtainable from turmeric; a second
ingredient
obtainable from green tea; and an acceptable aerosol spray vehicle. The
aerosol
composition may be administered as a treatment or prophylactically to birds
using any
conventional techniques for which an aerosol, spray or mist composition is
suitable,
e.g. spraying or misting of birds.
In a fourth aspect, the present invention relates to animal feeds, dry
formulations and liquid formulations which includes a first ingredient
obtainable from
turmeric and a second ingredient obtainable from green tea.
These and various other advantages and features of novelty that characterize
the invention are pointed out with particularity in the claims annexed hereto
and
forming a part hereof. However, for a better understanding of the invention,
its
advantages, and the objects obtained by its use, reference should be made to
the
accompanying descriptive matter, in which there is described, a preferred
embodiment
of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to a method for the prophylactic use of a composition to
reduce the incidence of contracting and/or transmitting Newcastle disease. The
method comprises the steps of administering to a bird that has been, or will
be,
exposed to the Newcastle virus, an amount of a composition having a first
ingredient
obtainable from turmeric; a second ingredient obtainable from green tea; and
an
acceptable carrier. The amount of anti-microbial composition is effective,
when
administered, to reduce the incidence of contracting and/or transmitting
Newcastle
disease. The composition may also be used to treat a bird infected with
Newcastle
disease by administering an effective amount of the composition to treat
Newcastle
disease.
The composition for use in the methods of the present invention may include a
first ingredient obtainable from turmeric, and a second ingredient obtainable
from
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,t...~~ ~.._, .. ~
green tea. Ingredients obtainable from ginger and horseradish may also be
included as
optional additional ingredients of the composition of the present invention.
As used herein, the term "acceptable" means a component that is suitable for
use with birds without undue adverse side effects (such as toxicity,
irritation, and
allergic responses), commensurate with a reasonable risk/benefit ratio.
Further, as used herein, the term "safe and effective amount" refers to the
quantity of a component, which is sufficient to yield a desired therapeutic
response
without undue adverse side effects (such as toxicity, irritation, or allergic
responses),
commensurate with a reasonable risk/benefit ratio when used in the manner
described
herein.
The term "inhibiting", as used herein, refers to reducing or preventing
further
growth of a Newcastle virus strain, or preventing the Newcastle viral strain
from
attaching to normal cells, and/or the elimination of some or all of the
infectious
particles from the human or animal being treated. Suitable methods for
determining
viral inhibition are discussed in the examples.
The term "transmissivity" or "transmitting" as used herein refers to the
transfer
of a microbe from one host to another.
All active compounds used in the present invention may be obtained from
other sources, if available. Thus, the phrase "which can be obtained from" or
the
phrase "which may be obtained from" is meant to encompass compounds or
compositions that are obtainable from turmeric, ginger, green tea or
horseradish, and
therefore encompasses synthetic forms of the same compounds and/or
compositions
as well as the same compounds and/or compositions obtained from other sources.
In a first embodiment, the composition of the present invention includes a
first
ingredient obtainable from turmeric, and a second ingredient obtainable from
green
tea, in a safe and effective amount to provide one or more of the beneficial
effects
described herein.
The first ingredient of the composition of the present invention may be
obtained from turmeric. The ingredient obtained from turmeric may be used in a
safe
and effective amount to provide one or more of the beneficial effects
described herein.
Turmeric (Curcuma longa), or Haldi in Hindi, is used very widely as medicine
as well
as a common ingredient in Indian cooking. The rhizome of turmeric is used in
medicine and food as a fine powder.
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The yellow pigment of the rhizome of turmeric is composed of three
compounds known as curcuminoids. The three curcuminoids are curcumin
(diferuloylmethane), desmethoxycurcumin (hydroxycinnamoyl feruloylmethane),
and
bis-desmethoxycurcumin (dihydroxydicinnamoyl methane) (see Drug Analysis,
Chromatography and Microscopy, p. 169, Ann Arbor Science Inc., 1973). The
essential oils of turmeric (Curcuma longa) are primarily composed of the
following
compounds: d-camphor (about 1%), cyclo-isoprenemyrcene (about 85%), and p-
tolylmethylcarbinol (about 5%), (see E. Gunther, The Essential Oil, pp. 123-4,
Van
Nostrand Co., 1955).
The ingredient of the composition of the present invention, obtained from
turmeric, preferably includes curcuminoids, such as curcumin
(diferuloylmethane),
desmethoxycurcumin (hydroxycinnamoyl feruloylmethane), and bis-
desmethoxycurcumin (dihydroxydicinnamoyl methane), and mixtures of two or more
of these curcuminoids.
Methods for isolating curcuminoids from turmeric are known (see Janaki and
Bose, An Improved Method for the Isolation of Curcumin From Turmeric, J.
Indian
Chem. Soc. 44: 985, 1967). Alternatively, curcuminoids for use in the present
invention can be prepared by synthetic methods.
The ingredient, which can be obtained from of turmeric, can be incorporated
into the composition of the present invention in a variety of different forms.
Those
different forms preferably include extracts of turmeric such as turmeric
extracts
including turmeric powder extracts, turmeric fluid extracts, Aquaresin
turmeric,
Oleoresin turmeric, one or more the curcuminoid compounds, and turmeric
powder,
parts of, or whole plants of turmeric, tinctures thereof, and mixtures
thereof. More
preferably, the first ingredient obtainable from turmeric is a turmeric
extract.
When the ingredient obtainable from turmeric is used, each gram of the
composition of the present invention preferably contains about 0.001 mg to
about 20
mg of an ingredient obtainable from turmeric such as turmeric powder extract.
Most
preferably, each gram of the compositions contains about 0.01 mg to about 1.5
mg of
an ingredient obtainable from turmeric such as turmeric powder extract. These
ranges
are based on the use of Turmeric Extract 95%, ex. Phaimline, Inc. in the
ingested
formulation and Turmeric Root Extract (Oleoresin(t Turmeric), ex. Kalsec,
Inc.,
Kalamazoo, Mich., in the spray formulation.
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The second ingredient of the composition of the present invention may be
obtained from green tea. The second ingredient obtained from green tea may
have an
antioxidant effect. Green tea is the dried leaves and leaf buds of the shrub
Camellia
sinensis. It is mainly produced in China and Japan. Dried tea leaves are
composed
mainly of phytochemicals known as polyphenols (about 36%), principally
flavonols
(including catechins), flavonoids, and flavondiols. The leaves also contain
plant
alkaloids (about 4%), including caffeine, theobromine and theophylline.
The phaimacological activities of green tea are mainly due to its active
compounds. The active compounds of green tea useful in the present invention
include, but are not limited to, flavonols, catechins, flavonoids,
flavondiols, plant
alkaloids, caffeine, theobromine, theophylline, phenolic acids, proteins,
carbohydrates, and minerals.
The second ingredient which may be obtained from green tea, can be included
in the composition in the form of green tea powder, green tea extracts such as
green
tea powder extracts, green tea fluid extracts, and one or more active
compounds of
green tea, part of, or whole green tea plants, green tea leaves, tinctures
thereof, or
mixtures thereof. Preferably, the second ingredient of the composition of the
present
invention is selected from green tea leaves, green tea powder and green tea
extract.
More preferably, the second ingredient of the composition of the present
invention is
green tea extract.
Each gram of the composition of the present invention preferably contains
about 0.001 mg to about 20 mg of an ingredient obtainable from green tea such
as
green tea extract. Most preferably, each gram of the composition contains
about 0.01
mg to about 15 mg of an ingredient obtainable from green tea such as green tea
extract. These ranges use, as a baseline, the use of Green Tea, ex. Stryker
Botanics in
the ingested formulation and Green Tea Extract, ex. Phytoway, Inc., ChangSha,
P.R.
China, in the spray formulation.
An optional ingredient of the composition of the present invention may be
obtained from ginger, and is used in a safe and effective amount. Native to
southern
Asia, ginger is a 2- to 4-foot perennial that produces grass-like leaves up to
a foot
long and almost an inch wide. Ginger root, as it is called in the grocery
store, actually
consists of the underground stem of the plant, with its bark-like outer
covering
scraped off.
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The active compounds of ginger which may be employed in the present
invention include, but are not limited to, 1,8-cineole, 10-dehydrogingerdione,
10-
gingerol, 6-gingerdione, 6-gingerol, 6-shogaol, 8-.beta.-l7-epoxy-.lambda.-
trans-12-
ene-15,16-diol, 8-gingerol, 8-shogaol, 9-oxo-nerolidol, acetaldehyde, acetic
acid,
alanine, .alpha.-linolenic-acid, .alpha.-linolenic acid, .alpha.-phellandrene,
.alpha.-
piene, .alpha.-terpinene, .alpha.-terpineol, .alpha.-zingiberene, ar-
curcumene,
arginine, ascorbic acid, asparagine, .beta.-bisabolol, .beta.-carotene, .beta.-
elemene,
.beta.-eudesmol, .beta.-ionone, .beta.-myrcene, .beta.-phellandrene, .beta.-
pinene,
.beta.-selinene, .beta.-sesquiphellandrene, .beta.-sitosterol, .beta.-thujone,
bornyl-
acetate, boron, caffeic acid, calcium, camphene, camphor, capric acid,
caprylic acid,
capsaicin, caryophyllene, chavicol, chlorogenic acid, chromium, citral,
citronellal,
citronellal, cobalt, copper, cumene, curcumin, cystine, delphinidin, .delta.-
cadinene,
elemol, ethyl acetate, ethyl-myristate, farnesal, farnesene, ferulic acid,
furfural,
.gamma.-aminobutyric acid, .gamma.-terpinene, geranial, geraniol, geranyl-
acetate,
gingerenone, glutamic acid, glycine, hexahydrocurcumin, histidine,
isogingerenone-B,
isoleucine, kaempferol, lecithin, limonene, linoleic acid, magnesium,
manganese,
methionine, mufa, myrecene, myricetin, myristic acid, neral, nerol, nerolidol,
niacin,
nickel, oleic acid, oxalic acid, p-coumaric acid, p-cymene, p-hydroxy-benzoic
acid,
palmitic acid, pantothenic acid, paradol, patchoulic alcohol, phenylalanine,
quercetin,
riboflavin, selenium, shikimic-acid, terpinen-4-ol, thiamin, tryptophan,
vanillic acid,
vanillin, zinc, and zingerone. Also, mixtures of two or more of these active
compounds may be employed.
Ginger, can be incorporated in the composition of the present invention in
many different forms including extracts such as ginger extracts including
ginger
powder extracts, ginger fluid extracts, ginger powder including ginger root
powder,
Aquaresin ginger, Oleoresin ginger, and one or more active compounds of
ginger,
parts of, or whole ginger plants, tinctures thereof, and mixtures thereof.
Preferably,
the optional ingredient of the composition of the present invention is
selected from
ginger extract and ginger powder.
Each gram of the composition of the present invention preferably contains
about 0.001 mg to about 30 mg of an ingredient obtainable from ginger such as
ginger
extract. Most preferably, each gram of the composition contains about 0.01 mg
to
about 20 mg of an ingredient obtainable from ginger such as ginger extract.
These
ranges use, as a baseline, the use of Ginger Root Powder, ex. Stryka Botanics
in the
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"C ..,t - -.,_, .. ._.
ingested formulation and Ginger Extract K(Aquaresin ginger), ex. Kalsec, Inc.
of
Kalamazoo, Mich. in the spray formulation.
The amounts of various ingredients are given herein in terms of one form of
the ingredient, i.e. ginger extract. If that ingredient is present in another
form, then the
amount to be employed is that amount which will provide the same amount of the
one
or more active compounds as the amount of that ingredient given herein.
Also, the composition of the present invention may include one or more
ingredients obtainable from horseradish, in a safe and effective amount to
provide one
or more of the beneficial effects described herein. The optional ingredient
obtainable
from horseradish may include extracts from the Cochlearia Armoracia and may be
in
the form of a horseradish extract, such as, for example, horseradish powder
extracts,
horseradish fluid extracts, and horseradish root extracts such as horseradish
oil.
Horseradish contains volatile oils that are similar to those found in mustard.
These
include glucosinolates (mustard oil glycosides), gluconasturtiin, and
sinigrin, which
yield allyl isothiocynate when broken down in the stomach. The compositions of
the
present invention preferably contain from about 0.0001 mg to 10 mg of an
ingredient
obtainable from horseradish such as horseradish oil per gram of the
composition, and
more preferably, from 0.001 mg to 5 mg of an ingredient obtainable from
horseradish
such as horseradish oil per gram of the composition.
Exemplary compositions of the present invention may include one or more
ingredients obtainable from each of turmeric and green tea; one or more
ingredients
obtainable from each of turmeric, green tea and ginger; one or more
ingredients
obtainable from each of turmeric, green tea and horseradish; or one or more
ingredients obtainable from each of turmeric, green tea, ginger and
horseradish.
2.5 Ethanol, propylene glycol and glycerin and various combinations thereof,
may
be optionally included in the liquid compositions of the present invention, up
to about
10 percent by weight of the total as an optional ingredients. Most preferably,
up to
about 10 percent per total weight ethanol is added as an optional ingredient.
Even
more preferable, 2.5 to 7 percent ethanol is added.
Pr-eferably, the main ingredients described above, that may be derived from
turmeric and green tea and, optionally, ginger and horseradish, make up from
about
0.001 to about 90% by weight of the total composition. More preferably, the
main
ingredients will make up about 0.01 to about 20% by weight of the total
composition.
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Most preferably, the main ingredients make up about 1 to about 10% by weight
of the
total composition
The non-carrier ingredients of the composition, including the ingredients
obtainable from turmeric, green tea, ginger and horseradish as discussed
above, can
be increased or decreased proportionally in the composition of the present
invention
depending on the amount of carrier used in the composition, without
substantially
affecting the effectiveness of the composition for its intended use.
The plant extracts, e.g., turmeric extract, ginger extract, green tea extract
and
horseradish extract that may be used in the compositions of the invention, may
be
produced using common extraction procedures. Alternatively, the extracts may
be
purchased from commercial sources such as the Kalsec, Inc. of Kalamazoo, Mich.
The processes for the preparation of pharmacologically or biologically active
plant extracts in a convenient, administrable dosage form from any of the
plants
mentioned above, are well known in the art.
The composition of the present invention may be used to prevent the
infectivity and transmissivity of various strains of the Newcastle disease
virus among
birds. The composition may also be used as a therapeutic composition to treat
Newcastle disease and symptoms associated with Newcastle disease.
A safe and effective amount of the composition of the present invention may
be administered to a bird that has been or will be exposed to Newcastle
disease, to
reduce the incidence of contracting said illness, relative to a bird that has
been or will
be exposed to the Newcastle disease virus.
Preferably, the composition of the present invention may be formulated in any
acceptable dosage for7n including, but not limited to animal feeds such as
bird feed,
powders, dry formulations of any type, liquid formulations of any type, such
as
sprays, suspensions, solutions, injections or any standard form for mass
inoculation.
The composition of the present invention may also be administered in the form
of a
nutritional supplement, in which case the composition of the invention may be
the
nutritional supplement or may form a part of a nutritional supplement
containing
additional ingredients.
Tablets in this invention may differ in shape, size and manufacturing
technique. In the case of tablets, for oral use, the acceptable carrier may
further
include lactose and corn starch. Lubricating agents may also be added to the
tablets,
including, for example, magnesium stearate, sodium lauryl sulfate and talc.
Tablets
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may also contain excipients such as sodium citrate, calcium carbonate and
calcium
phosphate. Disintegrants such as starch, alginic acid and complex silicates,
may also
be employed. Tablets may also include binding agents such as
polyvinylpyrrolidone,
gelatin, PEG-8000 and gum acacia.
Alternatively, the composition of the pr-esent invention may be formulated in
liquid form, such as syrups, solutions, liquid formulations, mists or sprays,
with a
solvent or dispersant such as water, or other liquids and optionally in a
pharmaceutically acceptable carrier, for repeated delivery of the composition
to oral
and oropharyngeal mucous membranes over a sustained period of time.
Preferably,
the treatment time is about 5 to 60 minutes, and more preferably about 20 to
30
minutes, so as to permit a prolonged contact of the composition with mouth,
nasopharnyx and throat tissues. Alternatively, such formulations can be in a
concentrated form suitable for dilution with water or other materials prior to
use.
The composition of the present invention may also be formulated with an
acceptable carrier. The acceptable carrier may include, but is not limited to:
(a)
glycerin; (b) ethanol; (c) phospholipids; (d) MCT oil; (e) water; and (f)
suitable
relatively insoluble excipients including starches, celluloses, cyclodextrins,
silicas and
lipids/fats.
The composition may also be formulated in chewable forms, such as animal
feeds, as a food additive or component of the animal feed. The composition of
the
invention may alternatively be formulated in capsule form, with or without
diluents.
For capsules, useful diluents include lactose and dried corn starch. When
suspensions
are employed, emulsifying and/or suspending agents may be employed in the
suspensions. In addition, solid compositions including one or more of the
ingredients
of the lozenges described above may be employed in soft and hard gelatin
capsules.
The composition of the present invention may also be formulated into an
aerosol or inhalant composition. Such a composition may be prepared using well-
known techniques. For these types of formulations, suitable carriers may
include the
following ingredients: saline with one or more preservatives, absorption
promoters to
enhance bioavailability, fluorocarbons, and/or conventional solubilizing or
dispersion
agents.
The composition may also be administered using any known standard delivery
means. The composition may be formulated as a water or solution additive.
Moreover,
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the composition may be administered in ovo. Optionally, the compositions of
the
invention can also be used as an anti-viral disinfectant for wiping down
surfaces.
Other materials, which may optionally be included in the composition of the
present invention, include resveratrol (trihydroxystilbene), inositol, other B-
complex
vitamins, and additional anti-inflammatories. Also, ingredients such as
sweeteners,
flavorants, coloring agents, dyes, preservatives, emulsifying agents,
suspending
agents, melting agents, excipients, demulcents and solvents or diluents such
as water,
ethanol, propylene glycol, glycerin and various combinations thereof, may be
included in the composition of the present invention.
Reducing or preventing transmission relates to preventing or reducing the
spread of a microbe from one bird (infected) to another bird (non-infected).
Some
birds may be considered carriers of the infection. Carriers are individuals
who
actively shed Newcastle microbes but do not suffer from an acute infection.
These
carriers may be said to be persistently (or chronically) infected with a viral
strain of
Newcastle. In addition to the persistently infected shedder, other infective
birds may
be those which are actively infected, and particularly those in the early or
late stages
of an acute infection. One aspect of the invention relates to administering to
a bird
infected with a strain of Newcastle disease, the composition of the present
invention,
to prevent the spread of the disease to other birds.
Prophylactic treatment is aimed at a bird that will soon be exposed to the
Newcastle virus or has recently been exposed to the Newcastle virus for the
purpose
of reducing the instance of active infection. Such prophylactic treatment may
be
effective either alone or in addition to a vaccine. The prophylactic treatment
of the
present invention may also be used against viral strains of Newcastle disease
for
which there is not yet a vaccine available.
The invention also relates to a method of treating a bird infected with
Newcastle disease to treat the disease by, for example, reducing the duration,
fatality
rate, or adverse effects of the disease. In another aspect of the invention,
the present
invention relates to a method for reducing, treating or at least partially
preventing of
at least one symptom or adverse effect of viral infection by administering, to
a bird
infected with the Newcastle virus, a composition of the present invention,
including
ingredients that can be obtained from ginger and green tea. Symptoms that may
be
treated include lack of energy, decreased egg production, soft shelled eggs,
swelling,
nasal discharge, coughing, gasping, head tremors, wing and leg paralysis,
twisted
12
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
,<-z - s .,_, .. ...
necks, impaired appetite, diarrhea, intestinal lesions, depression, loss of
appetite,
increased respiration and blue combs.
The composition of the present invention may be administered to any member
of the avian species, which includes the common commercial poultry birds:
chicken,
turkeys, ducks and geese, less commonly the ostrich as well as other bird
species that
are commonly kept as house pets, for example canaries and parrots.
The composition may be administered by directly spraying the composition
into the nasal passage of the bird, spraying the composition into the oral
cavity of the
bird or the composition may be administered by creating a mist to which the
birds are
exposed. Thus, the composition may be given prophylactically to act in a
virucidal or
virustatic manner. Alternatively, the composition may be used to reduce the
transmissivity of the virus.
The effective amount of the composition will vary depending on such factors
as the patient being treated, the particular mode of administration, the
activity of the
particular active ingredients employed, the age, bodyweight, general health,
sex and
diet of the bird, time of administration, rate of excretion, the particular
combination of
ingredients employed, the total content of the main ingredient of the
composition, and
the severity of the illness or symptom. It is within the purview of one of
ordinary skill
in the art to account for these factors.
The composition may be administered about 1 to about 15 times per day, as
needed, more preferably, about 2 to about 12 times per day, as needed, or most
preferably, about 6 to about 10 times per day, as needed. The composition of
the
present invention may be administered in any acceptable dosage form, as
described
above, including, but not limited to, tablets, capsules, powders, oral sprays,
nasal
sprays, nasal drops, chewable compositions, suspensions, solutions and through
in ovo
administration.
Each dosage of the composition contains a safe and effective amount of the
composition of the present invention. An effective amount for each therapeutic
administration contains a total of about 0.001 milligram to about 1 gram of
the
ingredients, which may be obtained from turmeric and green tea. More
preferably, an
effective amount of the composition for each therapeutic administration
contains a
total of about 0.01 milligrams to about 0.5 grams of the ingredients which may
be
obtained from turmeric and green tea. The amounts of the various ingredients
of the
13
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
1< --- s .._, .. -
composition administered in accordance with the method of the present
invention are
the same as given above for the composition of the present invention.
When the composition is administered as a feed or water additive, the amount
of the active ingredients in the feed or water additive may range from about
0.01 to 50
weight percent of the total feed composition. In a preferred embodiment, the
active
ingredients constitute about 0.1 to about 30 weight percent of the total feed
composition, and in a most preferred embodiment, the active ingredients
constitute
about 1 to about 20 weight percent of the total feed composition. The active
ingredients may comprise the ingredient from turmeric, the ingredient from
green tea,
the ingredient from ginger and/or the ingredient from horseradish.
When the composition is administered as a liquid, mist, spray, injection,
aerosol or mist, the amounts each of the active ingredients may be reduced as
the
spray composition delivers the active ingredients more directly to the
location where
they are needed, as compared to a lozenge or capsule for example. The
composition
may be diluted to any desired concentration with the addition of water or
another
suitable diluent; the diluted composition may contain anywhere from about 0.1%
to
about 99.999% by weight water or other diluent, more preferably about 55% to
about
95% water or other diluent by weight and most preferably 70% to about 90%
water or
other diluent by weight.
The following preferred ranges define compositions according to the invention
that are suited for administration in a liquid formulation, such as a spray,
according to
the methods of the invention.
Each gram of the composition administered in a spray according to the
methods of the present invention preferably contains about 0.001 mg to about
12 mg
of a turmeric extract such as soluble or miscible oleoresin turmeric. Most
preferably,
each gram of the composition contains about 0.01 mg to about 9 mg of a
turmeric
extract such as soluble or miscible oleoresin turmeric. Each gram of the
composition
administered in a spray according to the methods of the present invention
preferably
contains about 0.001 mg to about 20 mg of a green tea extract such as green
tea leaf
extract. Most preferably, each gram of the composition contains about 0.01 mg
to
about 15 mg of a green tea extract such as green tea leaf extract.
Each gram of an optional embodiment of a composition administered in a
spray according to the methods of present invention optionally contains about
0.001
mg to about 10 mg of a ginger extract such as Aquaresin ginger. Most
preferably,
14
CA 02668933 2009-05-07
WO 2008/074028 P,CT/US2007/087536
AZ viv-ivri = =
each gram of the composition contains about 0.01 mg to about 7 mg of a ginger
extract such as Aquaresin ginger.
Optionally, each gram of the composition also contains from about 0.0001 mg
to about 5 mg of horseradish root extract, more preferably about 0.001 mg to
about 2
mg and most preferably about 0.5 mg to about 1 mg of a horseradish root
extract.
An effective amount of the composition may also be used to disinfect and/or
sterilize any equipment used to administer the composition to the birds so as
to
inactivate some or all of any strain of the Newcastle disease virus located on
the
equipment. The composition may be topically applied to any equipment or
machine
surface to disinfect the instrument.
The invention will be further illustrated by the examples given below which
are not to be construed as limiting the invention in any way. The scope of the
invention is to be determined by the claims appended hereto.
Example 1
A Suitable Formulation - Composition 1
Component Target (wt %) Target (g) Actual (wt g) Actual (wt %)
Turmeric 0.6466 0.6466 0.6952 0.6943
Oleoresin
Ginger 0.6840 0.6840 0.6826 0.6817
Oleoresin
Horseradish Oil 0.063120 0.0631 0.0631 0.0630
Green tea, 0.4619 0.4619 0.4646 0.,4640
powered extract
Glycerin 46.5723 46.5723 46.6322 46.5701
Ethanol 5.0000 5.0000 5.0157 5.0090
Phospholipids 0.5000 0.5000 0.5093 0.5086
MCT oil 5.0000 5.0000 5.0033 4.9966
Water 41.0721 41.0721 41.0673 41.0126
Total 99.9981 100.0000 100.1333 100.0000
This formulation may be diluted by a factor of 1-1300 with water or another
suitable
diluent to provide more dilute compositions for use in a variety of
applications such as
spraying, misting, as an aerosol or as a liquid formulation.
Example 2
A study of the safety and tolerability of Composition 1 to chickens using
various dosages and routes of administration was performed. The results
demonstrated that Composition 1 is an effective and suitable liquid additive
to poultiy
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
.~~-- --.., .. .-.
water supply, liquid additive to a nasal drop formulation or solid additive to
poultry
feed.
132 White Leghorn chickens, approximately 7 days old, were separated into
11 groups, administered various forms and concentrations of Composition 1
corresponding to Tables 1(a)-1(c) and subsequently exposed to the Newcastle
virrus.
Table 1(a)-1(c) summarize the dosage, route of administration and
concentrations of a composition in accordance with the invention given to 132
White
Leghorn chickens in a tolerability and toxicity study.
Table 1(a)
Chicken
Group No. of Chickens Numbers Routn of Administration Dose Concentrafion
1 12 1- 12 feed (continuous) high
2 12 13 - 24 feed (continuous) med
3 12 25 36 feed (continuous) low
4 12 37 - 48 water (continuous) high
5 12 49 - 60 water (continuous) med
---- 61 - 72 ----- - water (continuous) low
-- _ 6 12
--
7 12 73 - 84 feed & water (continuous) high
8 12 85 - 96 feed & water (continuous) med
9 12 97 - 108 feed & water (cont_inuous) low
10 12 109 - 120 control (none) -
11 12 121 - 132 nasal drop -
Spare bird supply 12 n/a n/a -
Total Birds 144
Table 1(b)
Dos-_.
Greuo F~i;~~ cf i^irislratlon (-o'~cert'al-c[I C~sinp_] Lr.~ii C) i
1 feed (continuous) high 1- 4 ad libitum
2 feed (continuous) med 1 4 ad libitum
3 feed (cont_inuous) low 1-4 ad libitum
4 water (continuous) high 1- 4 ad libitum
5 water (continuous) med 1- 4 ad libitum
6 water (continuous) low 1-4 ad_ libitum
7 feed & water (continuous) high 1- 4 ad libitum
8 feed & water (continuous) med 1- 4 ad libitum
9 feed & water (continuous) low 1-4 ad_libitum
10 control (none) - 1- 4 ad libiturn
11 r asal drop - 1- 4 1 drop per nostril 4 X day
Spares n/a - none none
16
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
O
-j O oCDCD CO0 CJ(D
C:') =
a 3 .~
g, ~=
ooo~d
o
O O O O O' O
O O O O O O~I.
!
o 0 0 0 0 0 0 0 0 0
s ~~'~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~ ~ ~=i
0 1--
a w_
p~ N NO O O N N N O O J
(Do
.
CD
O O C D ~ O O O
O O C O O
( . _...._ .........
W
~
D~~
06 06 06
~~~~
- - - -
~>
Z~
(~, r- N ch d' Lo Cfl I-- c0 m'~. O~ I~ C
17
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
~lJill-Ill( ~ .. iJ
The chickens were housed three in each cage. The cages were maintained at
about 85 degrees Fahrenheit and with a relative humidity at 65%. They were
provided
with 16 hours of continuous, incandescent lighting, followed by 8 hours of
darkness
each day.
The chickens were monitored to determine their tolerability to and the
toxicity
of Composition 1. Quantities of water and feed consumed by each group were
assessed and individual chicken weights were routinely weighed and measured.
For chickens which were administered Composition 1 in the form of nasal
drops, the nasal drops were administered, one (1) drop per each nostril, four
(4) times
daily, for 4 days of dosing, in alignment with the feed and water dosing
groups.
Drops may be administered twice in the morning with each administration being
approximately 1 hour apart, and twice in the afternoon/evening also with each
administration being approximately 1 hour apart. This nasal dosing schedule is
flexible so that a total of 4 drops per each nostril can be administered per
day, yet
consecutive morning or afternoon doses are not spaced closer than 1 hour
apart. Nasal
drops were administered using a standard bottle type dropper containing 20
drops / ml,
or 50 microliters per drop.
For chickens which were administered Composition 1 as a feed additive, the
feed additive was provided for a 4 day period. One group of 24 chickens was
given a
high dose of the feed additive. Another group of 24 chickens were given a
medium
dose, and another group of 24 chickens were given a low dose. Feed and water
was
provided ad libitum for 4 days, under routine conditions.
For chickens which were administered Composition 1 as a water additive, the
water additive was provided for a 4 day period. One group of 24 chickens were
given
a high dose of the water additive. Another group of 24 chickens were given a
medium
dose, and another group of 24 chickens were given a low dose. Water was
provided to
the chickens ad libitum for 4 days, under routine conditions. Feed was
provided ad
libitum. The control group in this experiment were housed and fed according to
standard conditions for 4 days.
To deter7nine the optimal effective dosage, each group of chickens were
administered Composition 1 for a period of 1-4 days out of the week. The
chickens
were observed daily for any signs of general malaise or abnormal behavior, to
include
ruffled feathers, depressed posture, down birds, or any other indicators of
stress or
discomfort. Any lesions or abnormalities noted were recorded a daily basis. At
the
18
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
end of the study, the chickens were euthanized and individually necropsied to
search
for any lesions. Collected samples were placed in 10% formalin for
histological
assessment.
Feed and water were weighed and measured out on a per cage and per day
basis. Amounts were be recorded on data collection forms. On Day 1 feed and
water
was measured and recorded for the first time. On Days 2-4, additional feed and
water
was measured and documented as added to the feed and water listing. On Day 5,
the
remaining feed and water were weighed, and the total feed and water consumed
on a
per cage basis for the entire study period was calculated.
Table 2 summarizes the change in body weight data. The differences among
the treatment groups were not statistically significant for chickens which
were given
low feed, low water, low feed and water, medium feed, high feed, nasal and
control.
19
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 2
Body leleig-nt 4rsr r~1 5tmiary
Peot 18 L r' 1 tr-d
'Pre- Post- Charsge:
Group Statist:ic '~~~E, -7= - - Treatmerit Past-Pre
Control N SIRI7S 9 9
1 ; .1 39.1 a.
1FIAN a7.0
ME-IrN 88..7 1 ^ ~, 38.2
S^D 5.2 :.~ 2.6
T~'N '~9,8 113.9 357
MAX 54.6 135.3 4'?.6
P--VUTIE 0.= C i i il
E~eed t; FI_ I 12 12 12
1iE. P1 85.8 122.6 5~=3 a
1lE=iI:1R 85.0 119.5 36, 1
8.8 11.3 4.9
Td=2~ 67.1 99.3 27.8
pLA~ 97.5 139.0 43.7
P-VALUE 0.3000
High Feed a_:,_i 7.6- .-.,_,r N BI-RDS 12 12 12
MEAN = . 88.3 -2.1 C
MEDIAN ~ 9. 4 90.7 -0.8
^ D_ _ . 2 12.9 12.2
Ti-11 ? N. 4 -1 7. 7
Pti~~f 1=ir,2 11 .- 23.4
P-VAL,ta'E 47 s5536
High Watex N BIRDS 12 12 12
ME.-W 8; 6 .. 5 7.1 b
MEDIAN 8.7.4 { 1,' 10.0
STD 7.6 22.2
Mild 69.2 .:3 -V 7{ . 9
TLu" 9-11.6 141.9 4 ~4.17
p - -;r:._L r; E o. ~_ 15
Low Feed N BURDS 12 12 12
MEAN 92.7 1316. 7 44.0 a
14E77IAN 93.5 135 , 6 43.5
STD 12.4 5.1
M-TN 111.3 35.4
MAX 159.0 52.8
P-SIALUE 0.0000
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
~V1V-1Vi1/ =v
Table 2 (continued)
Ba>dIr ~ L~~f' t (91-aw) S'Luma.rY
Pr.-_ 18 De3.ete-d
P-e_ ~:, = Ch,H__rTle
~~eatmex.t, Trea=:epn` T r~ atTten.t P= -.-P:~e
Low Feed and Water N BIRDS 12 12 1.2
MEAN ~4.4 121.3 36.9 a
I~~I IAN 117,9 35.0
10.5 5.5
9.1 108.2 30.8
P~l~{ 96.3 142.9 47.4
F Sr-E 0.9000
Low ~~ar-er ti BI=.L s 12 12 1.2
131.7 41.4 a
iiE Irl< ~ 130.5 41.3
? ^_ _ 9.1 13.5 5.5
M=Yd 74.5 112.7 32.4
M~-a 1015,2 160.0 54.8
P - E 0.0000
Medi1~ Feed C; E'I==I s 12 12 12
ME:-:tT 86.8 127.5 40,7 a
IriEDII~,: 88.1 129.4 40.9
8,6 1ia4 4.8
;A--D
11=tI 71.2 108.5 31.4
K-_x 103.6 143.0 49.8
g '-ALr-E 0.9000
Medium Feed and Water N B I.L 12 1.2 1.2
2+LE=211 =! r:. , :5 78.2 - 8. 4 C
ME IAN ?_. u 70.4 -14 . 8
S--U 13.9
14_AZ 5, ? 58.6 -21.3
ALu: 1U _'.5 12ua4 18.9
g ;Lf'E Ct. G,61:3
Medi.tm Ttdatr~.r N E' I=L s 12 12 12
191~21 84.6 ? 4 a 1. -1 0. 5 C
MEDI.tT 83.8 72.7 -13.3
8.0 J,~ 11.5
111It~ 74.2 -22. 6
pi:-L,`r 97,3 : f 2. 6 1 a.5
I'-'vFA.LT-T~', 0.9089
21
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
~. ~~. ~.,.., .. ..
Table 2 (continued)
Body Sseigixt (grams) S-imtaar}r
Peii 18 Deleted
Pre- Post- r_`hange:
Treat.mexit Group Statistic Treatm?sit Treatment Frst-Fre
Nasal Drop N BIRDS 12 12 12
IMEA.IN 84 , 6 122 A 37.7 a
NSSEDIAN 87..7 123,6 37.5
STD 11..5 16.2 6.1
MIN 69.2 94.6 30 .0
R3FiX 97.7 14 6. 8 49,1
P-VALTJE 0.0000
OVERALL P-VALUE FOR fiRF,ATMENT GROUP C:024PARISrJLd0.314:5 <0.. 0C1(}1
Table 3 summarizes the water and feed consumption data. In this table the data
are summarized on a per pen basis. 4 cages that were not used were included in
the
analysis to show the naturally occurring feed and water loss. The differences
among
the treatment groups were not statistically significant for chickens which
were given
low feed, low water, low feed and water, medium feed, high feed, nasal and
control
groups.
22
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 3
Feed and Water Constimption Siuma~~
Pen 118 Deleted
Feed Water
Treatment Cogi~iimption Cons-Lunp-tickri
Group Statistic (tjrams) (MI-S)
CAGE NOT USED N PENS 4 4
~EAN 1.8 d 203.8 a
MEDIAN 1.5 189.5
L,TL; 1.4 30.9
MIN 0.5 186.0
MAX 3.7 250.0
Control N PENS 3 3
MEAN 1,94: . .3 a 434. O a
raETaIAN 192.1 1 427.0
;=.TL:, 4.3 26.2
MIN 191.6 412e 0
MAX 199.3 463.0
Hi-crI7 F & W N PENS 4 4
PEAN 78.4 bc 296.0 b
MT- LiIAN 77.9 289.5
ST.La 40.7 44.7
MIN 38.9 253 . 0
MAX 118.8 8 352.0
I-Figli Feed ANl I'EN ~,t 4 4
ME.Z%-N 19 i=i . 4 a 417.8 a
hELIIAN 1.;;;. _ 458.0
2
STD 17.3 92.2
MIN 172.1 280.0
MAX 211.7 475 . 0
High 14ater N PENS 4 4
blEAN 9:7. 9 b 262.0 bo
MEDIAN 98.0 270.5
TL i 63.8 47.6
hiIN 27.7 202.0
K~:?i 167. 9 305. O
23
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
~V1V-1Vlrl =V
Table 3 (continued)
Feed and Water Co1 z,ff:ptio~~ S-Lmma~y
Pen 18 De4.eted
Feed Water
Treatment Conslum~~ion t:,nnsi_atnrition
Group Statistic (grams) (mls)
I,owF &W N 1'E4 4
bfEA21 183.0 ~.~ ~ 38 . 8 a
MF-DIAN 178.9 4-~6. :S
STD 9.1 40.0
MIN 177.5 387A
mzox 196.5 475.0
Lr:*,,= Feed N 4 4
j.E.A--r,j 197. 1 a 437.8 a
MEDIAN 195.3 441.5
STD 9.9 21.5
MII.~ 187.0 410.0
Low Water N F'EP;; Q 4 4
I-1EA24 194.9 a 418.3 a
MEDIAN 191.1 4'~~.1 . .,~s
STD 8.3 43..4
MIN 190.0 365A
MAX 207.3 465.0
Meditim F W N PEI4S 4 4
ME.++N 49.6 c 277. 0 b
hEI?II-14 47.8 284.5
STD 16.3 39..1
MIN 33.7 228.0
MAX 69.1 311.0
Medi-tim Feed N PENS 4 4
NiEAN 191.8 a 447.3 a
MEDIAN 197.3 4:57.0
ST"D 18.2 32.7
MIN 165.8 400.0
MAX 206.8 475.0
24
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 3 (continued)
E`eer.~ and Water Consumption S=ary
Pen 18 Deleted
Faedi Water
Treatment canstunption C-'ensiimption
Group Statistic (grams) {mls}
Medium Water N PENS 4 4
M-EAti 51. 9 a 2 62 . 3br.
MEi7IAN 52. 8 271..5
STD 7. fl 29,0
MIN 42,9 220 . 0
MAX 59.3 285 , 0
Nasal Dsops N PENS 4 4
I-IEAId 1 81 . 6 a 427. 5 a
MEDZ.AN 186.5 435.5
STD 12.1 36.1
MIN 163.8 382 , 0
MAX 189,4 4:5'7,0
OVERALL P-VALUE FOR 1'REAT14El`F'P GROUP COMPARISON <0, 0001 <0. 0001
Table 4 summarizes the ratio of weight gain to feed consumption data. In this
table, the data are summarized on a per pen basis. The analysis revealed that
the
differences among the following treatment groups were not statistically
significant:
low feed, low water, low feed and water, medium feed, high feed, nasal and
control.
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 4
Body Weight (grams) / Feed Cons-ur:ptlon :~~~~~ ~~ra:rs~
Pen 18 Delete;a
E, (_ ,: ly
Tre,-, tment. W 1 g11 r/F~ ~d
Gr~.~~.a~ ~~atisti_c
CoIitz:_ l N LENS 3
t IEJ ~~ ~1 O. ~ ~ 3 a
ME DIKN 0.600
STD; OA1 f,
C~ ~ +
MIN
AIAX
P-VALT_TE 0 2
High F W N FELIS 4
MEryI - O.CF; 6 .
11EUIT-S -(=E .:1s9
STLi 0. 56R
MI N - 0. 6 ;
1' -VAL,rrE U. 6303
Higl3 Feed I~;' F'ENS 4
P=IEAT-T 0.582 a
~~I 0.582
ED.I~:~'
STD O. i; ; V~
bdIAI 0.523
MAX 0.640
P-'VAIJYE 0.0002
High Water I.~ FIENS 4
t=iE1~_0d' - 0. 254 ~.?
P=SED .IAK i i .1919
S'L"L} 1.181
MItN -2. 0,=i 4
Y-Ax 0.590
P-VAi<TTE 0,6963
26
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 4 (continued)
Body Weight (ga-ams) / Feed Con~~~~~~~~ S-Lum~.~~~ (grams)
Pen ~~ Deleted
P t_;:aTr,.
Treatment ~Te i c-T 1-i t % Fe e :1
Gro,_11-1 ~tati .-~i,_! uons,_urq-,t.Ton
Low F & W N PENS 4
MFAN 0.604 a
MEDIAN 0.. 622
0.044
MIN 0.539
MAX 0! . 633
F-VA-LUE 0.0001
Low Feed N PENS 4
NiEAN ~.~~9 a
MELaI~T 0 . 670
STD 0.025
MIN O. r ; ~ ~
MAX
P-VA-I.rYE 0. 1- i : a 1 O
Low Water N F' Eld S. 4
MEAN 0.638 a
NIF-1~ IAN 0.638
STD 1_1. C108
MIN 627
MAX _i . 64 6
P-VATuTTE 0.0000
~ieditim. F ~ W N PEtl ~ 4
I+= -i_1.7iJ3 b
NfEDIAN -0.778
STD 0.843
MIN -1 . 4 ~4
MAX 0.197
P-VA.LLE 0.1916
27
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
Table 4 (continued)
4l:it (grams) :7~7_:.d i=`~ansurp.tior~ ~-Lurtmary (gran~)
L~~~~~ ~~ic
ii;n 18 Delef_.e;_1
Body
T'reatrLt:-:~n+- ~Te iglit- ,f'Feed
t"ro'Li~ ~~~~~stic C:_:ns-ulia :,t.ion
Medium Feed 4
~=~~ ~ 0.636 a
t=iEDI : ,.Ii 0 . 630
SZD 0.027
bdI t.1 0.610
MAX 0.674
P -'VA-LUE 0 . 0000
Medium. W-ater N FE14;73 4
ME.P41N - 0. i_e 5`=3 1.=
I3EDI AN - '~ . ~ '_~ `_=~
S TL3 I. 17,5 F=~
PrIIN - 1. 41 15
MAX -0 .~11_~
P-VALUE 0 0 9 ; F,
Nasal Frrc~Fs I, PENS 4
MEZLIT ~J. ~ ~ 23 a
MED I 111I U. f-~ 2 9
'=!.'TLi 0. U15
t~tTN 0.600
MAX 0.634
P-VALUE 0.0000
These results provide suitable dosing for Composition 1 for poultry. The data
demonstrates that Composition 1 can be provided to growing poultry in a
medicated
feed at various (low, medium, and high) concentrations, without safety issues
or
tolerability problems.
Example 3
The undiluted antiviral composition of Example 1, Composition diluted in 10-
fold serial dilutions were prepared and tested for antiviral activity against
NDV in
VERO E6 cells and in 10-day old embryonating chicken eggs. In addition, a
placebo
was similarly diluted and tested.
28
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
The continuous cell line VERO E6 (CRL-1586) was obtained from the
American Type Culture Collection (Rockdale, MD) and propagated in Minimum
Essential Medium (Eagle) with 2mM L-glutamine, 1.5g/L sodium bicarbonate, 0.1
mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% fetal bovine
serum
(Invitrogen Corp [Gibco], Carlsbad, CA) at 37C and 5% COZ. The cells were
grown
in a T75 flask (BD Biosciences, Franklin Lakes, NJ) and transferred to 96 well
plates
and grown to 90% confluence.
A 1x103 concentration of B1/BI strain of NDV, a tissue culture infectious
dose50 (TCID50), was mixed with seven 10-fold serial dilutions (beginning with
a
dilution of Ix10-3, which is nontoxic for the cells) of the antiviral compound
or the
placebo prepared in cell culture maintenance media (containing 1% fetal bovine
serum). The mixtures were incubated at room temperature for 30 minutes; then
nine
10-fold serial dilutions of each mixture were prepared for inoculation onto
cells. The
cell culture media was removed from the cells and the mixtures were inoculated
onto
the monolayers. Negative control wells receiving cell culture maintenance
media only
were also included in the experiment. The cells were incubated for 7 days at
37C and
5% CO2 and examined twice daily for cytopathic effects (CPE).
Fig. 1 shows the results of the antiviral affect on NDV. Fig. 3 shows VERO
E6 cells protected from viral infection by the composition of the present
invention and
cells, to which the composition had not been applied, infected with NDV viral
CPE. A
dilution of Ix10-3 reduced the TCID50 titer of NDV 5-fold and a 1 X 10-4
dilution of
the composition reduced the NDV titer 1.8-fold. None of the higher dilutions
of the
composition reduced the titer of NDV. By comparison, no reduction in virus
titer for
NDV was observed for the placebo at any dilution, which suggests that the
composition was responsible for the antiviral effect. Additionally, none of
the
negative control wells had CPE.
Embryonic chicken eggs, infected with NDV, were also tested to determine
the efficacy of the composition. The embryonic chicken eggs were prepared in
the
same fashion as that of the VERO E6 cells. The experimental design was the
same,
except 10-day old embryonating chicken eggs were inoculated instead of tissue
culture cells and the beginning concentration of Composition 3 and the placebo
was
undiluted since the compounds are not toxic for the embryos. Specific pathogen
free
(SPF) fertile chicken eggs were obtained from Sunrise farms (Catskill, NY) and
incubated at 37C for 10 days. The embryonated eggs were inoculated into the
29
CA 02668933 2009-05-07
WO 2008/074028 PCT/US2007/087536
"IL .,z ._. -- , .. ._.
chorioallantoic sac (CAS) with 200ul of undiluted and each of the 10-fold
dilutions of
the composition or the placebo prepared in PBS (pH 7.4) and mixed with either
1 X
104 embryo infectious dose50 (EID50) of 1 X 107 EID50 of NDV. Negative control
eggs
that received PBS only were also included. The eggs were incubated at 37C and
candled daily for 7 days to record mortality. Any mortality occurring within
the first
24 hours was considered to be due to trauma associated with inoculation and
disregarded. On the 7th day, all the remaining eggs were chilled to 4C and
opened to
examine the embryos for clinical signs.
Fig. 2 shows a 100-fold reduction in titer with the undiluted composition
whereas less than a 100-fold reduction was observed for dilutions of the
composition
from 1 X 10-1 to 1 X 10-5 and only a slight reduction was observed at a 1 X
10"6
dilution of the composition. Similar to the VERO E6 cells, the placebo did not
reduce
the titer of either virus in embryonating eggs, indicating that the active
ingredient in
the composition was responsible for the antiviral effect. Thus, Composition 1
appears
to have measurable affects against NDV at low and high concentrations.
It is to be understood, however, that even though numerous characteristics and
advantages of the present invention have been set forth in the foregoing
description,
together with details of the structure and function of the invention, the
disclosure is
illustrative only, and changes may be made in detail, especially in matters of
shape,
size and arrangement of parts within the principles of the invention to the
full extent
indicated by the broad general meaning of the terms in which the appended
claims are
expressed.
